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Comparative Study
, 26 (8), 2094-103

A Novel Human AP Endonuclease With Conserved Zinc-Finger-Like Motifs Involved in DNA Strand Break Responses

Affiliations
Comparative Study

A Novel Human AP Endonuclease With Conserved Zinc-Finger-Like Motifs Involved in DNA Strand Break Responses

Shin-ichiro Kanno et al. EMBO J.

Abstract

DNA damage causes genome instability and cell death, but many of the cellular responses to DNA damage still remain elusive. We here report a human protein, PALF (PNK and APTX-like FHA protein), with an FHA (forkhead-associated) domain and novel zinc-finger-like CYR (cysteine-tyrosine-arginine) motifs that are involved in responses to DNA damage. We found that the CYR motif is widely distributed among DNA repair proteins of higher eukaryotes, and that PALF, as well as a Drosophila protein with tandem CYR motifs, has endo- and exonuclease activities against abasic site and other types of base damage. PALF accumulates rapidly at single-strand breaks in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner in human cells. Indeed, PALF interacts directly with PARP1 and is required for its activation and for cellular resistance to methyl-methane sulfonate. PALF also interacts directly with KU86, LIGASEIV and phosphorylated XRCC4 proteins and possesses endo/exonuclease activity at protruding DNA ends. Various treatments that produce double-strand breaks induce formation of PALF foci, which fully coincide with gammaH2AX foci. Thus, PALF and the CYR motif may play important roles in DNA repair of higher eukaryotes.

Figures

Figure 1
Figure 1
PALF and distribution of CYR motif in eukaryotes. (A) Amino-acid sequence alignment of FHA domains of human PALF and human PNK. (B) Proteins with CYR motif. Domains and number of amino acids in proteins with tandem CYR motifs (tandem type), PALF (human) and CG6171-PA (D. melanogaster) and with a single copy of the motif (single type), Glaikit (Tdp1) protein (D. melanogaster), DNA ligase III (C. elegans) and uracil DNA glycosylase (Dictyostelium discoideum) are shown. (C) Sequence alignment and conserved amino-acid residues in CYR motifs. CYR1 and CYR2 are the first and the second motifs in human PALF as shown in (B).
Figure 2
Figure 2
Nicking activity of PALF against various types of DNA damage. (A) Silver staining of His-tagged PALF (His-PALF) purified from BL21 host E. coli cells. (B) Nicking activity of His-PALF against various types of damaged base on one strand in 30-mer double-stranded DNA. fAP (AP site with furan ring), Tg (thymine glycol), 8-oxoG (8-oxoguanine), uracil, HOU (5-hydroxyuracil), HMU (5-hydroxymethyluracil), OHC (5-hydroxycytosine), DHT (5,6-dihydrothymine) and Etheno-A (1, N6-ethenoadenine) are located at the 14th base of the 5′ 32P-labeled 30-mer strand, which was treated with His-tagged PALF. Two arrows in fAP lane show the major nicked sites. (C) Purified His-PALF after gel filtration shown by silver staining (upper panel) and nicking activity of fractions 10, 12 and 14 against AP site (lower panel). (D) Nicking activity of GST-tagged PALF (GST-PALF) and GST alone at the fAP site. Recombinant proteins were prepared from E. coli PRC501 strain and purified simply through a glutathione Sepharose column. (E) Nicking activity of GST-PALF purified through glutathione Sepharose, HiTrap Heparin, HiTrap Q and HiTrap S columns. Nicking activity against fAP with EDTA or Mg2+ (left) or against HOU (right) is shown. (F) Nicking activity of His-PALF against an AP site compared with that of human AP endonuclease (APE1) in sequencing gel. (G) Determination of nicked sites using 3′-end-labeled oligos with an AP site. Oligos were treated with His-APE1 or His-PALF with or without subsequent CIP phosphatase treatment. (H) Nicking activity of His-PALF against 5-hydroxyuracil (HOU) is compared with nicking activity of NEIL1 and fAP digested by His-PALF shown on sequencing gel.
Figure 3
Figure 3
Nicking activity of CYR domain of hPALF and Drosophila CG6171-PA against various types of DNA damage. (A) Purified His-tagged CYR domain (His-CYR from amino-acid residues 361–551, Coomassie blue staining) of PALF. (B) Nicking activity of His-CYR of PALF against various substrates. Abbreviations are the same as in (B). (C) Purified His-tagged Drosophila CG6171-PA (His-CG6171) shown by Coomassie blue staining. (D) Nicking activity of His-CG6171 against various substrates. Abbreviations are the same as in (B). (E) Nicking activity of His-CG6171 against an AP site in sequencing gel. Nicking activity is compared with that of APE1. (F) Determination of nicked site by 3′-end-labeled oligos containing an AP site. Oligos were treated with APE1 or His-CG6171 with or without subsequent CIP phosphatase treatment, which partially removed the phosphate from the substrates.
Figure 4
Figure 4
Accumulation of PALF at locally produced DNA damage. (A) Accumulation of endogenous PALF after irradiation with laser. PALF (left indicated with arrows) and XRCC1 (middle) were identified with antibodies and were merged together (right) in HeLa cells after 250 scans of 405-nm laser light. (B) Accumulation of GFP-PALF at sites of laser micro-irradiation. Immediately after one pulse of 365-nm laser light to a HeLa cell through an F20 filter (left before and middle after irradiation) or five scans of 405-nm laser light (right), GFP-tagged PALF has accumulated at the irradiated site as indicated by the arrows. (C) Accumulation of PALF at UVDE-induced SSBs in XPA-UVDE cell. After UV irradiation of XPA-UVDE cell through a porous filter, the spots of accumulated GFP-PALF (second from left) are merged (far right) with those of XRCC1 (third from left) stained with antibody. No SSB was produced in XPA-vector cells (far left). (D) Enzymatic activity of GFP-PALF. Purified GFP-PALF (left panel) shows AP endonuclease activity (right). (E) Accumulation and dissociation kinetics of GFP-PALF after 5 scans of 405-nm laser light in mouse cells. Intensity of fluorescence caused by accumulated GFP-PALF is measured in mouse wild-type cells (squares), wild-type cells treated with PARP inhibitor DIQ (diamonds) and PARP1 KO cells (circles). Data presented are the mean values of six independent experiments. (F) Minimum accumulation domain of PALF at SSB. GFP was tagged to the amino terminus of each construct. + and – indicate accumulation or no accumulation, respectively.
Figure 5
Figure 5
Physical and functional interactions of PALF with PARP1. (A) Direct interaction of PARP1 with PALF. Four domains of PARP1 and GST-tagged regions are shown in the upper figure. GST-tagged full-length PARP1 as well as its zinc-finger domain are able to pull down His-PALF (middle panel). GST-tagged full-length PALF and the CYR domain (see Figure 4F) pull down His-PARP1 (bottom panel). ΔFΔC domain is the region between FHA and CYR domains (Figure 4F). (B) Poly(ADP) ribosylation of PALF and PARP1 by PARP1. Salmon testis DNA (0.2 μg) was sonicated and added to the reaction. Transfer of 32P from NAD to each of the proteins was identified by autoradiography. Recombinant His-PARP1 is cleaved during its preparation from E. coli cells. (C) PALF activates PARP1 in the presence of plasmids harboring SSBs. Plasmids prepared by UV irradiation and UVDE treatment were mixed without (three left-hand panels) or with His-PALF (three right-hand panels) and ribosylation of PARP1, POLβ and Histone H1 by PARP1 was measured by autoradiography of 32P transferred from NAD to the proteins. (D) PALF but not APE1 activates PARP1 at AP sites. Plasmids treated with glyoxal were mixed with APE1 (two left-hand panels) or His-PALF (two right-hand panels) and ribosylation of PARP1 and POLβ was measured. (E) Influence of knock-down of PALF expression in HeLa cells on MMS sensitivity. Colony-forming ability after MMS treatment of HeLa cells transfected with siRNA designed to suppress PALF expression is shown. Mean values of three independent cell survival experiments for siPALF1 and siPALF2 (open circles) and control (open squares) are shown. Expression of PALF was determined by Western blotting using anti-PALF antibody (β-actin as loading control).
Figure 6
Figure 6
Involvement of PALF in DSB responses. (A) Putative PALF-binding proteins identified by affinity chromatography and mass spectrometry. His-PALF was attached to a nickel agarose column for binding of proteins prepared from HeLa nuclear extracts. Control lane shows the proteins obtained by column attached with a control protein (GST). All the proteins identified so far by this method are shown. (B) GST-tagged KU86 and LIGASEIV, but not KU70, XLF and XRCC4, are able to pull down His-PALF. (C) GST-XRCC4 phosphorylated either by CK-II or by DNA-PKcs pulls down His-PALF (upper panels); the FHA domain of PALF is pulled down more effectively by phophorylated GST-XRCC4 (lower panels). (D) GST-PALF and GST-tagged domain, ΔΦΔX, between FHA and CYR domains pull down His-tagged KU86. (E) Nicking activities of PALF against various double-strand ends. Three substrates used for analysis of the nicking activity of PALF at DSBs are shown (left). Stars indicate the sites for 32P-labeling. Nicked DNA fragments were identified in sequencing gel after treatment of the three substrates (right). Determined nicked sites are indicated by arrows on the left. (F) Formation of PALF foci after treatment of HeLa cells with various agents. Cells were treated with X-rays (6 Gy) and hydroxyurea (HU, 10 μM); cells were treated with anti-PALF antibody 30 min after X-ray treatment or 2 h after HU treatment. (G) Colocalization of PALF and γH2AX foci induced by CPT treatment of HeLa cells. Two hours after 2 μM CPT treatment, cells were double-stained with antibodies against PALF and γH2AX.

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